INTRODUCTION

The following protocol describes the synthesis of short linear peptides, or peptide pools, on modified cellulose membranes, and the detection of their protein-binding partners. Peptides are synthesized from their carboxyl termini using Fmoc-amino acid derivatives. After completion of the synthesis and cleavage of all side-chain-protecting groups, the peptide array on the membrane is incubated with the potential interaction partners to identify their target sequences.

DMF should be free of contaminating amines and thus of the highest affordable purity (e.g., peptide synthesis grade from Biosolve BV). Purity can be checked by adding 10 μl of bromophenol blue indicator stock solution to 1 ml of DMF. If the resulting color is yellow, the batch can be used without further purification. Be sure to check each new batch.

Ethanol, technical grade (95%) (optional; see Step 12)

Fmoc-AA stock solutions

Fmoc-amino acid (Fmoc-AA) derivatives of all 20 L-amino acids as well as β-alanine and other special amino acid derivatives are available from several suppliers at sufficient quality (Novabiochem/Merck Biosciences or Bachem). The side-chain-protecting groups should be Cys(Acm) or Cys(Trt), Asp(OtBu), Glu(OtBu), His(Trt), Lys(Boc), Asn(Trt), Gln(Trt), Arg(Pmc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). HOBt-esters of these amino acid derivatives must be prepared in NMP for use throughout in spotting reactions. Dissolve 1 mmol of each Fmoc-AA in 5 ml of NMP containing 0.25 M HOBt to give 0.2 M Fmoc-AA stock solutions. These are kept in 10-ml plastic tubes that are closed tightly, flash-frozen in liquid nitrogen, and stored at -70°C. For use in coupling reactions with amino acid mixtures at randomized positions in the peptide sequences, combine equal aliquots of Fmoc-AA stock solutions for the respective amino acids to be incorporated, dilute with twofold volume of NMP to give 66 mM solutions, and store as described above.

Horseradish peroxidase (HRP)-conjugated detection antibodies

N-Hydroxybenzotriazole (HOBt), anhydrous (ISOCHEM)

Store tightly closed at room temperature in a dry place.

Immun-Star Chemiluminescent Kit (No. 170-5018, Bio-Rad)

Membrane blocking solution (MBS)

Methanol, technical grade (95%) (optional; see Step 12)

1-Methyl-2-pyrrolidinone (NMP)

For use in the preparation of Fmoc-AA stock solutions, NMP should be of the highest purity available. Amine contamination can be checked by adding 10 μl of bromophenol blue indicator stock solution to 1 ml of NMP. If the resulting color is yellow, the NMP can be used without further purification. Most commercial products, however, are not acceptable. To purify an unsuitable batch, treat 1 liter of NMP with 100 g of acid aluminum oxide overnight under constant vigorous shaking at room temperature. The next day, retest the purity; a 1-ml aliquot should give a yellow BPB test. Filter the slurry through a bed of dry silica gel (for flash chromatography, Mallinckrodt Baker BV) in a closed glass filter funnel (slight nitrogen pressure can speed up the process, but is not necessary). Divide the clear liquid into 100-ml portions and store tightly closed at -20°C.

Phosphate-buffered saline (PBS)

Piperidine mix

Special chemical derivatives

Free thiol functions of cysteine may be problematic because of post-synthetic uncontrolled oxidation. To avoid this, Cys may be replaced by Ser, Ala, or α-aminobutyric acid (Abu). Alternatively, choose the hydrophilic Cys(Acm) and leave protected. For the simultaneous preparation of peptides of different size with free amino termini, couple their terminal amino acid residues as αN-Boc derivatives so that they will not become acetylated during the normal elongation cycle. Boc is then removed during the final side-chain de-protection procedure.

Stripping mix A (SM-A)

Stripping mix B (SM-B)

Transfer buffer for western blotting

Tris-buffered saline (TBS) without phenol red

T-TBS (TBS buffer plus 0.05% Tween 20)

Tween 20

Equipment

All equipment used for membrane synthesis and regeneration should be resistant to organic solvents. Glassware or polypropyleneware should be exclusively used in all steps involving organic solvents. Standard micropipetting tips (Gilson, Eppendorf) can be employed.

3MM paper (Whatman)

Blotting apparatus (e.g., Biometra-Fast-Blot)

Digital recording device

Scanner or CCD camera for documentation of signal patterns on membranes plus analysis software for quantification of signals. In case of radioactive or chemiluminescence detection, autoradiography films can be used.

Dispensers, adjustable from 5 ml to 50 ml for DMF and alcohol containers

Flat glass tray to hold at least one membrane

Flat reaction/washing troughs with a tightly closing lid made of chemically inert material (glass, Teflon, polypropylene) with dimensions slightly larger than the membranes used

AC-S01 type amino-PEGylated membranes (AIMS-Scientific-Products GmbH) are recommended and available from several suppliers (Hartmann Analytic GmbH; Rapp Polymere GmbH; Intavis AG). Ready-to-use membranes in an 8 × 12 format with 96 spots of βAla anchors are available from Sigma-Genosys.

The ASP222 instrument requires a special format of membrane with perforations for the holder pins on the robot. These are available only from Intavis AG

SPOT synthesis kit (Sigma-Genosys)

Software for the generation of peptide lists and pipetting protocols is included in the Sigma-Genosys synthesis kit and the operation software of the spotting robot. A freeware package is also available from the authors ([email protected]).

METHOD

All volumes given below are for one standard AC-S01 paper sheet of 8 × 12 cm and must be adjusted for more sheets, or other paper qualities and sizes. Unless otherwise stated, washes and incubation steps are carried out in sealed troughs at room temperature with gentle agitation on a rocking platform. Solvents and solutions are decanted after the time indicated. During incubations and washes, the troughs are closed with a lid.

Stage 1: Preparative Work

Generate a list of peptides to be prepared. Multiple lists can be combined on a single membrane to fill up a complete array. The peptides can be separated after synthesis by simply cutting the membrane into appropriate sections.

Cutting lines between sections can be marked out on the membrane in pencil.

Select the appropriate array(s) for the chosen experiment according to number, spot size, and scale required.

Calculate the required volumes of Fmoc-AA solutions for each derivative and cycle.

Remember that a triple coupling procedure may be necessary, and that each vial should contain a minimum of 50 μl. Consider, for example, a list of peptides that requires alanine for 26 peptides at cycle 1 on a 17 × 25 array. This cycle will require 26 (peptides) × 0.2 (μl per elongation) × 3 (couplings) = 15.6 μl of Fmoc-Ala stock solution. Therefore, you will take the minimum of 70 μl of stock solution for this vial. The SPOT software can perform this calculation.

Label a set of 1.5-ml microfuge tubes with derivative and cycle code (e.g., A1), and distribute the Fmoc-amino acid stock solutions according to the calculated volumes required. Snap-freeze in liquid nitrogen and store at -70°C.

Stage 2: Generation of the SPOT Array

5.Mark each membrane used with a pencil label (e.g., a number or letter at the right bottom edge) for proper orientation and tracking throughout the synthesis process. For manual synthesis, mark the spot positions on the membranes with pencil dots, and place the membrane in the reaction trough. For automated synthesis, fix membranes on the platform of the spot robot.

6.Take a 100-µl aliquot of the Fmoc-βAla stock from the freezer and bring it to room temperature. Add 1 μl of BPB stock and 4 μl of DIC. Mix, and leave for 30 minutes. Spot aliquots (0.5 μl for 8 × 12 array, or 0.1 μl for 17 × 25 array) of this solution on all positions of the chosen array configuration. Cover the membrane with glass plates, and allow the reaction to proceed for 60 minutes.

For peptides longer than 20-mers, reduce the loading of the spots by applying a mixture of the Fmoc-βAla stock and an N-acetyl-alanine stock (1:9). This will avoid molecular crowding.

7.Wash each membrane twice in 20 ml of acetylation mix; once for 30 seconds, and once for 2 minutes. Incubate the membranes overnight in acetylation mix.

Spots should be stained only light blue! If traces of remaining piperidine on the membranes turn the liquid dark blue, renew the staining solution and continue the staining.

12.Wash each membrane in 20 ml of methanol or ethanol (two times for 10 min each).

13.Transfer the membranes to 3MM paper folders, and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C.

Stage 3: Assembly of Peptides on SPOTs

14.Take the membranes from Step 13 and, for manual synthesis, number the blue spot positions with a pencil (according to the peptide lists). Place the membranes in separate reaction troughs. For automated systems, fix the unmarked membranes on the platform of the synthesizer. If necessary, cutting lines should be marked in pencil. If bound protein is to be eluted from individual spot positions after probing, these should also be marked.

15.Take the appropriate set of Fmoc-amino acid stock aliquots for cycle 1 from the freezer, bring to room temperature, and activate by adding DIC (4 μl per 100-μl vial; ~0.25 M). Incubate for 30 minutes at room temperature. For manual experiments, pipette aliquots of the Fmoc-amino acid solutions onto the appropriate spots on the membrane. For automated experiments, place the vials containing the activated Fmoc-AA solutions into the rack of the spotting robot, and start cycle 1. Leave for 15 minutes. Repeat the spotting twice, and allow the reaction to proceed for 2 hours (cover the membranes on the spotter with glass plates).

Monitor the amino acid coupling reaction by inspection of the spot color change. Spots should turn yellow-green during this step. If some spots remain dark blue, additional applications of Fmoc-amino acid stock solution can be made.

i. For the introduction of randomized X positions in the peptide sequences, take the appropriate Fmoc-AA-mix stock solution from the freezer, bring to room temperature, and activate by adding DIC (1.5 μl per 100 μl-vial; ~0.09 M). Incubate at room temperature for 30 minutes. Perform spotting four times.

16.Wash each membrane twice with 20 ml of acetylation mix (once for 30 seconds, and twice for 2 min). Incubate the membranes in fresh acetylation mix for ~10 minutes (until all remaining blue color has disappeared).

Again, the spots should be stained only light blue. If traces of remaining piperidine on the membranes turn the liquid dark blue, replace the solution and continue the staining. BPB staining is charge-specific. Therefore, it does not only bind to amino-terminal amino groups. The side chains and protecting groups of the amino acids in the peptide chain can strongly influence staining intensity. The visible color of the peptides depends on the overall charge and, therefore, on the individual amino acid sequence.

21.Wash each membrane with 20 ml of methanol or ethanol (two times for 5 min each).

22.Transfer the membranes to 3MM paper folders, and dry them using cold air from a hair dryer.

23.Repeat this procedure from Steps 15 to 23 for successive elongation cycles.

Stage 4: Terminal Acetylation

Synthetic peptides mimicking fragments of a longer continuous protein chain should be amino-terminally acetylated to avoid the production of an artificially charged terminus. To accomplish this, continue as follows after the final amino acid elongation cycle from the stage above:

24.Incubate each membrane in 20 ml of acetylation mix for a minimum of 30 minutes (until all remaining blue color has disappeared).

25.Wash the membranes in 20 ml of DMF (three times for 2 min each), and then in 20 ml of alcohol (two times for 5 min each).

26.Transfer the membranes to 3MM paper folders, and dry them using cold air from a hair dryer.

Stage 5: Side-Chain De-protection

After the peptide assembly is complete, all side-chain-protecting groups are removed from the peptides. Trifluoroacetic acid is extremely hazardous; the following procedure must be performed in a chemical fume hood.

27.Prepare 40 ml of de-protection mix.

28.Place the dried membrane in the reaction trough, add de-protection mix, close the trough very tightly, and incubate overnight with gentle agitation.

This harsh treatment is required for complete cleavage of protecting groups. Cellulose membranes less resistant than AC-S will not survive this treatment.

29.Subject each membrane to the following series of washes:

i. 20 ml of DCM (four times for 5 min each)

ii. 20 ml of DMF (three times for 5 min each)

iii. 20 ml of alcohol (three times for 5 min each)

iv. 20 ml of acetic acid (1 M in H2O) (three times for 5 min each)

Wash each membrane with 20 ml of alcohol (three times).

30.Transfer the membranes to 3MM paper folders, and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C, or process further as described in the next section.

Stage 6: Protein-Binding Assay

This basic procedure has been optimized for use with AP-conjugated detection antibody and a color signal development. As mentioned above, horseradish peroxidase-labeled detection agents require the use of hydrogen peroxide, which gradually destroys the peptides on the array. More sensitive detection can be achieved with a chemiluminescent substrate of AP (e.g., Immun-Star). If such a substrate is used, follow the manufacturer’s instructions for Steps ix to xi of Method A. Alternatively, test proteins can be labeled prior to incubation with the peptide array by chemical biotinylation and subsequently detected using AP-conjugated streptavidin (under the same conditions as for AP secondary antibody). If fluorescent or radioactive labeled reagents are used, adapt Steps v to xi of Method A accordingly.

IMPORTANT: If using Method A, prior to probing your protein with the peptide spots on the membrane, always do a “pre-run” in which you first apply this protocol while omitting Steps v and vi. This is necessary to control for unspecific signals from components of the detection process or remaining proteins from a previous experiment on the same membrane. However, in case the proteins are electro-transferred and detected on a secondary nitrocellulose membrane (Method B, below), this precaution does not apply.

31.Perform protein-binding assay using either Method A or Method B.

Method A

i. Place a single membrane in a polystyrene plate, and wet it with a few drops of methanol or ethanol.

This is to enhance rehydration of any peptide spots that might be hydrophobic. The peptide locations should not be visible as white spots! If this happens, extend the alcohol treatment in a sonication bath at room temperature until spots have disappeared.

ii. Wash the membrane in 10 ml of TBS (three times for 10 min each).

iii. Incubate overnight in 10 ml of MBS.

The blocking conditions can be critical to the success of an experiment, and, depending on the protein of interest, it may be necessary to try several different blocking solutions to optimize the signal-to-noise ratio. The following solutions represent increasingly stringent blocking conditions: (1) 2% (w/v) skim milk powder in TBS; (2) 2% (w/v) skim milk powder, 0.2% (v/v) Tween 20 in TBS; (3) MBS; (4) MBS with 50% (v/v) horse serum. Blocking solution 3 is recommended for most applications.

iv. Wash the membrane once in 10 ml of T-TBS for 10 minutes.

v. Incubate for 2-4 hours in the presence of probe antibody (or protein) diluted in 8-10 ml of MBS.

For monoclonal antibodies, or pure proteins, use ~4-5 μg of purified antibody per milliliter of incubation volume. When using a polyclonal serum, we recommend a dilution of 1:100. It is not necessary to use a large volume of protein solution for the incubation. However, make sure that the membrane is completely covered throughout the incubation. To prevent drying out, use a lid, or seal the membrane in a plastic bag.

vi. Wash the membrane three times in 10 ml of T-TBS (for 10 min each).

viii. Wash the membrane two times in 10 ml of T-TBS (for 10 min each).

ix. Wash the membrane two times in 10 ml of CBS (for 10 min each).

x. Transfer the membrane to a flat glass tray and add 10 ml of CDS. Incubate without agitation until good signals are obtained.

For individual peptides on spots, this usually takes 10-30 minutes; peptide pools may require longer incubations (2 hr to overnight).

xi. Stop the reaction by washing the membrane twice in PBS. Keep the membrane wet, either in PBS or covered in plastic wrap. Store at 4°C.

The picture of signals on the membrane can now be documented by photography or (better) be electronically digitized with a scanner. A high-quality electronic image can be used to quantify signal intensities. Avoid drying of the membrane at this stage. If the membrane dries out, proteins may denature and become difficult to remove. After successful documentation of signals by photography or electronic scanning, continue with membrane stripping (see Steps 32-35).

Method B

If only weak binding of the test protein is anticipated, or if excessive background was observed in Method A, it may help to electro-transfer the bound test protein onto a secondary nitrocellulose membrane and repeat the detection procedure. Here, any detection system appropriate for nitrocellulose can be used (e.g., HRP conjugates), as the peptides will not be affected.

i. Place a single membrane in a polystyrene plate, and wet it with a few drops of methanol or ethanol.

This is to enhance rehydration of any peptide spots that might be hydrophobic. The peptide locations should not be visible as white spots! If this happens, extend the alcohol treatment in a sonication bath at room temperature until spots have disappeared.

ii. Wash the membrane in 10 ml of TBS (three times for 10 min each).

iii. Incubate overnight in 10 ml of MBS.

The blocking conditions can be critical to the success of an experiment, and, depending on the protein of interest, it may be necessary to try several different blocking solutions to optimize the signal-to-noise ratio. The following solutions represent increasingly stringent blocking conditions: (1) 2% (w/v) skim milk powder in TBS; (2) 2% (w/v) skim milk powder, 0.2% (v/v) Tween 20 in TBS; (3) MBS; (4) MBS with 50% (v/v) horse serum. Blocking solution 3 is recommended for most applications.

iv. Wash the membrane once in 10 ml of T-TBS for 10 minutes.

v. Incubate for 2-4 hours in the presence of probe antibody (or protein) diluted in 8-10 ml of MBS.

For monoclonal antibodies, or pure proteins, use ~4-5 μg of purified antibody per milliliter of incubation volume. When using a polyclonal serum, we recommend a dilution of 1:100. It is not necessary to use a large volume of protein solution for the incubation. However, make sure that the membrane is completely covered throughout the incubation. To prevent drying out, use a lid, or seal the membrane in a plastic bag.

vi. Wash the membrane three times in 10 ml of T-TBS (for 10 min each).

vii. Briefly equilibrate the peptide membrane and a sheet of nitrocellulose, trimmed to fit the peptide membrane, in transfer buffer.

viii. Electro-transfer the proteins bound to the peptide spot membranes onto the nitrocellulose membrane for 1 hour at 0.85 mA/cm2. IMPORTANT: Owing to SDS denaturation, all proteins will be negatively charged. Therefore, the nitrocellulose should be placed toward the positive electrode.

Depending on the chemical properties of the protein ligands, the time required for the transfer might differ and, therefore, must be determined empirically.

ix. Block the nitrocellulose membrane with MBS for 2 hours at room temperature.

x. Incubate the nitrocellulose membrane for 75 minutes with an AP- or HRP-conjugated detection antibody, or AP-/HRP-streptavidin for biotinylated proteins diluted in MBS.

Use dilutions comparable to those employed in immunoblots after SDS-PAGE.

xi. Wash the nitrocellulose membranes three times for 5-10 minutes each in T-TBS, and then three times for 5-10 minutes each in TBS.

To avoid damage to the adsorbed protein, do not wipe or press tissue onto the membrane.

xiii. Detect the spots using a chemiluminescence detection kit according to the manufacturer’s instructions. Be sure to include a positive control for the kit.

If no signal has been detected after 30 minutes of exposure, check that the positive control has worked, and repeat the experiment using less stringent blocking. If problems persist, this may indicate a discontinuous binding site or very low affinity binding.

In case of nonspecific signals or high background, increase the stringency of the blocking conditions, and make sure that the primary binding partner and detection reagent (e.g., antibody) are of high purity and are used in the highest possible dilution.

Stage 7: Membrane Stripping

A peptide spot membrane that was used in a protein-binding assay can be stripped off bound protein and reused for multiple-protein binding assays. In principle, thanks to the stability of the immobilized peptides, membranes can be regenerated up to 50 times without loss of signal intensity.

In some cases, proteins can resist removal from the spots, and those membranes can be used once only for Method A (on-spot membrane detection). Remains of bound protein must be checked by probing a stripped spot membrane first with the detection system (see above, Stage 6, Protein-Binding Assay protocol). Alternatively, Method B of Stage 6 can be applied.

Use the following procedure to strip the membrane.

32.Wash the spot membrane two times for 10 minutes each in 20 ml of H2O.

33.Incubate in 20 ml of DMF, until the blue color of spot signals has dissolved (usually ~10 min; incubate in a sonication bath at 40°C if necessary). Remove the solution and wash once more for 10 minutes in 20 ml of DMF.

This step can be omitted if a detection method other than a dye precipitation or Method B of Stage 6 was used.

34.Subject the membrane to the following series of washes:

i. 20 ml of H2O (three times for 10 min each)

ii. 20 ml of SM-A in a sonication bath at 40°C (three times for 10 min each)

iii. 20 ml of SM-B (three times for 10 min each)

iv. 20 ml of alcohol (three times for 10 min each)

35.Start the new binding assay at Step 31, Substep ii of the protein-binding assay protocol of Stage 6, or transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C.